Substituted condensation products of N-benzyl-3-indenylacentamides with heterocyclic aldehydes for neoplasia

ABSTRACT

Substituted condensation products of N-benzyl-3-indenylacetamides with heterocyclic aldehydes are useful for inducing or promoting apotosis and for arresting uncontrolled neoplastic cell proliferation, and are specifically useful in the arresting and treatment of neoplasias, including precancerous and cancerous lesions.

This application is a Continuation of prior U.S. application Ser. No.09/741,970 filed on Dec. 20, 2000 entitled “Substituted CondensationProducts of N-Benzyl-3-Indenylacetamides with Heterocyclic Aldehydes forNeoplasia,” now U.S. Pat. No. 6,426,349, which is a Continuation of Ser.No. 09/490,269 filed Jan. 24, 2000 entitled “Substituted CondensationProducts of N-Benzyl-3-Indenylacetamides with Heterocyclic Aldehydes forNeoplasia,” now U.S. Pat. No. 6,166,053 which is a Continuation of priorU.S. application Ser. No. 09/206,245 filed Dec. 7, 1998 entitled“Substituted Condensation Products of N-Benzyl-3-Indenylacetamides withHeterocyclic Aldehydes for Neoplasia,” now U.S. Pat. No. 6,066,634,which is a Continuation-in-Part of prior U.S. application Ser. No.08/989,353 filed Dec. 12, 1997 entitled “Substituted CondensationProducts of N-Benzyl-3-Indenylacetamides with Heterocyclic Aldehydes forNeoplasia,” now U.S. Pat. No. 5,948,779, all of which are incorporatedherein by reference.

TECHNICAL FIELD

This invention relates to compounds and methods for inducing orpromoting apotosis and for arresting uncontrolled neoplastic cellproliferation, methods that are specifically useful in the arresting andtreatment of neoplasias, including precancerous and cancerous lesions.

BACKGROUND OF THE INVENTION

Pharmaceuticals that are effective against early stage neoplasiascomprise an emerging and expanding area of research and potentialcommercial development. Such pharmaceuticals can delay or arrestdevelopment of precancerous lesions into cancers. Each year in theUnited States alone, untold numbers of people develop precancerouslesions, which exhibit a strong statistically significant tendency todevelop into malignant tumors, or cancer. Such lesions include lesionsof the breast (that can develop into breast cancer), lesions of the skin(that can develop into malignant melanoma or basal cell carcinoma),colonic adenomatous polyps (that can develop into colon cancer),cervical dysplasia (cervical cancer) and other such neoplasms.

Compounds that prevent or induce the remission of existing precancerousor cancerous lesions, or carcinomas, delay the onset of cancer and wouldgreatly reduce illness and death from at least certain forms of thatdisease.

Such compounds and methods are particularly beneficial tosub-populations of patients who repeatedly develop precancerous lesions,and therefore have a statistically higher probability of getting cancer.Many cancer types (e.g., breast, colon, prostate etc.) have such patientsub-populations. One example of a sub-population that will invariablydevelop cancer (if left untreated) includes those patients who sufferfrom familial polyposis of the colon. Familial polyposis patientstypically develop many (e.g., hundreds or thousands) of colonic polypsbeginning in their teenage years. Because each colonic polyp (whetherfamilial or non-familial) reportedly has approximately a five percentlifetime risk of developing into a cancer, the treatment of choice—untilvery recently—for familial polyposis patients is surgical removal of thecolon in the early twenties.

Many other cancers have sub-populations that also have much higher risksfor getting cancer at an early age and for having the cancer reoccur,than patients as a whole who get such a cancer. For example, suchsub-populations have been identified among breast cancer patients andcolon cancer patients. In the latter sub-population, removal of theindividual polyps as they form is the current treatment of choice.Removal of polyps in non-familial patients has been accomplished eitherwith surgery or fiber-optic endoscopic polypectomy—procedures that areuncomfortable, costly (the cost of a single polypectomy ranges between$1,000 and $1,500 for endoscopic treatment and more for surgery), andinvolve a small but significant risk of colon perforation.

The search for drugs useful for treating and preventing neoplasias intheir earliest stages is intensive because chemotherapy and surgery oncancer itself is often not effective, and current chemotherapy hassevere side effects. Thus, the search for compounds effective againstprecancerous lesions without the side effects of conventionalchemotherapy is particularly intensive. Such compounds are alsoenvisaged for recovered cancer patients who retain a risk of cancerreoccurrence, and even for cancer patients who would benefit fromcompounds that selectively induce apoptosis in neoplastic, butsubstantially not in normal cells.

Standard cancer chemotherapeutic drugs are not considered appropriatedrugs for cancer chemoprevention because whatever cancer preventative(as opposed to cancer-fighting) capabilities those drugs may possess donot outweigh their severe side effects. Most standard chemotherapeuticsare now believed to kill cancer cells by inducing apoptosis (alsosometimes referred to as “programmed cell death”). Apoptosis naturallyoccurs in virtually all tissues of the body. Apoptosis plays a criticalrole in tissue homeostasis, that is, it ensures that the number of newcells produced are correspondingly offset by an equal number of cellsthat die. Apoptosis is especially pronounced in self-renewing tissuessuch as bone marrow, immune cells, gut, and skin. For example, the cellsin the intestinal lining divide so rapidly that the body must eliminatecells after only three days to protect and prevent the overgrowth of theintestinal lining.

Standard chemotherapeutics promote apoptosis not only in cancer cells,but also in normal human tissues, and therefore have a particularlysevere effect on cells that normally divide rapidly in the body (e.g.hair, gut and skin). The results of those effects on normal cellsinclude hair loss, weight loss, vomiting and bone marrow immunesuppression. This is one reason standard chemotherapeutics areinappropriate for cancer prevention.

In the absence of a one-time cure (e.g., a gene therapy), another reasonis that cancer prevention therapy requires chronic administration of apharmaceutical to repress neoplasia formation, which for standardchemotherapeutics is obviously contraindicated because of the types ofside effects discussed above.

Abnormalities in apoptosis can lead to the formation of precancerouslesions and carcinomas. Also, recent research indicates that defects inapoptosis play a major role in other diseases in addition to cancer.Consequently, compounds that modulate apoptosis could be used in theprevention or control of cancer, as well as other diseases.

Several non-steroidal anti-inflammatory drugs (“NSAIDs”), originallydeveloped to treat arthritis, have shown effectiveness in inhibiting andeliminating colonic polyps. Polyps virtually disappear when the patientstake the drug, particularly when the NSAID sulindac is administered.However, the continued prophylactic use of currently available NSAIDs,even in polyposis syndrome patients, is still marked by severe sidereactions that include gastrointestinal irritations, perforations,ulcerations and kidney toxicity believed to be produced by inhibition ofprostaglandin synthetase activity (“PGE-2”). Such inhibition is arequirement for the NSAIDs anti-inflammatory action since elevatedlevels of PGE-2 are associated with inflammation. PGE-2 plays aprotective function in the gastrointestinal tract, which is the reasonsuch gastric side effects arise with chronic NSAID therapy, which israrely indicated for arthritis sufferers, acute therapy being the normfor them. However, chronic administration of sulindac is important forpolyposis patients to eliminate and prevent future polyps which causesgastric side effects in many such patients. Once NSAID treatment isterminated due to such complications, the polyps return, particularly inpolyposis syndrome patients.

Compounds such as those disclosed in U.S. Pat. No. 5,643,959 haveexhibited advantages in the treatment of neoplastic lesions since suchcompounds have been shown to induce apotosis in neoplastic cells but notin normal cells in humans. Thus, the severe side effects due toinduction of apotosis in normal cells by conventional chemotherapeuticsare avoided by these novel therapeutics (see, “Phase I Trial of SulindacSulfone in Patients With Familial Polyposis (FAP) With Rectal Polyps:Optimal Dose and Safety,” Digestive Disease Week, Abstract No. 2457, May10-16, 1997, American Gastroenterological Association et al.). Inaddition, such compounds do not exhibit the gastric side effectsassociated with NSAIDs since such compounds do not substantially inhibitPGE-2. More potent compounds with such neoplasia specificity but withoutsubstantial PGE-2 activity are desirable.

SUMMARY OF THE INVENTION

This invention represents potent compounds, that induce apotosis inneoplastic cells (but not substantially in normal cells), for treatingpatients with neoplastic lesions without substantially inhibiting PGE-2.This invention also involves methods for inducing such specific apotosisin neoplastic cells by exposing such cells to a pharmacologicallyeffective amount of those compounds described below to a patient in needof such treatment. Such compositions are effective in modulatingapoptosis and modulating the growth of precancerous lesions andneoplasms, but are not suffering from the side effects of conventionalchemotherapeutics and NSAIDs.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, the present invention includes compounds of FormulaI below (as well as their pharmaceutically acceptable salts) fortreating a patient with neoplastic, particularly precancerous, lesions:

wherein R₁ is independently selected in each instance from the groupconsisting of hydrogen, halogen, lower alkyl, lower alkoxy, amino, loweralkylamino, di-lower alkylamino, lower alkylmercapto, lower alkylsulfonyl, cyano, carboxamide, carboxylic acid, mercapto, sulfonic acid,xanthate and hydroxy;

R₂ is selected from the group consisting of hydrogen and lower alkyl;

R₃ is selected from the group consisting of hydrogen, halogen, amino,hydroxy, lower alkyl amino, and di-loweralkylamino;

R₄ is hydrogen, or R₃ and R₄ together are oxygen;

R₅ and R₆ are independently selected from the group consisting ofhydrogen, lower alkyl, hydroxy-substituted lower alkyl, amino loweralkyl, lower alkylamino-lower alkyl, lower alkyl amino di-lower alkyl,lower alkyl nitrile, —CO₂H, —C(O)NH₂, and a C₂ to C₆ amino acid;

R₇ is independently selected in each instance from the group consistingof hydrogen, amino lower alkyl, lower alkoxy, lower alkyl, hydroxy,amino, lower alkyl amino, di-lower alkyl amino, halogen, —CO₂H, —SO₃H,—SO₂NH₂, and —SO₂(lower alkyl);

m and n are integers from 0 to 3 independently selected from oneanother;

Y is selected from the group consisting of quinolinyl, isoquinolinyl,pyridinyl, pyrimidinyl, pyrazinyl, imidazolyl, indolyl, benzimidazolyl,triazinyl, tetrazolyl, thiophenyl, furanyl, thiazolyl, pyrazolyl, orpyrrolyl, or substituted variants thereof wherein the substituents areone or two selected from the group consisting of halogen, lower alkyl,lower alkoxy, amino, lower alkylamino, di-lower alkylamino, hydroxy,—SO₂(lower alkyl) and —SO₂NH₂.

Preferred compounds of this invention for use with the methods describedherein include those of Formula I where:

R₁ is selected from the group consisting of halogen, lower alkoxy,amino, hydroxy, lower alkylamino and di-loweralkylamino, preferablyhalogen, lower alkoxy, amino and hydroxy;

R₂ is lower alkyl;

R₃ is selected from the group consisting of hydrogen, halogen, hydroxy,amino, lower alkylamino and di-loweralkylamino, preferably, hydrogen,hydroxy and lower alkylamino;

R₅ and R₆ are independently selected from the group consisting ofhydrogen, hydroxy-substituted lower alkyl, amino lower alkyl, loweralkylamino-lower alkyl, lower alkyl amino di-lower alkyl, —CO₂H,—C(O)NH₂; preferably hydrogen, hydroxy-substituted lower alkyl, loweralkyl amino di-lower alkyl, —CO₂H, and —C(O)NH₂;

R₇ is independently selected in each instance from the group consistingof hydrogen, lower alkoxy, hydroxy, amino, lower alkyl amino, di-loweralkyl amino, halogen, —CO₂H, —SO₃H, —SO₂NH₂, and —SO₂(lower alkyl);preferably hydrogen, lower alkoxy, hydroxy, amino, amino lower alkyl,halogen, —CO₂H, —SO₃H, —SO₂NH₂, and —SO₂(lower alkyl);

Preferably, at least one of the R₇ substituents is para- orortho-located; most preferably ortho-located;

Y is selected from the group consisting of quinolinyl, isoquinolinyl,pyridinyl, pyrimidinyl and pyrazinyl or said substituted variantsthereof.

Preferably, the substituents on Y are one or two selected from the groupconsisting of lower alkoxy, amino, lower alkylamino, di-loweralkylamino, hydroxy, —SO₂(lower alkyl) and —SO₂NH₂; most preferablylower alkoxy, di-lower alkylamino, hydroxy, —SO₂(lower alkyl) and—SO₂NH₂.

The present invention also is a method of treating a patient with suchlesions by administering to a patient a pharmacologically effectiveamount of a pharmaceutical composition that includes a compound ofFormula I, wherein R₁ through R₇ and Y are as defined above. Preferably,this composition is administered without therapeutic amounts of anNSAID.

The present invention is also a method of treating individuals withneoplastic lesions by administering a pharmacologically effective amountof an enterically coated pharmaceutical composition that includescompounds of this invention.

Also, the present invention is a method of inhibiting the growth ofneoplastic cells by exposing the cells to an effective amount ofcompounds of Formula I, wherein R₁ through R₇ and Y are defined asabove.

In still another form, the invention is a method of inducing apoptosisin human cells by exposing those cells to an effective amount ofcompounds of Formula I, wherein R₁ through R₇ and Y are defined as abovewhere such cells are sensitive to these compounds.

Additionally, in yet another form, the invention is a method of treatinga patient having a disease which would benefit from regulation ofapoptosis by treating the patient with an effective amount of compoundsof Formula I, wherein R₁ through R₈ are defined as above. The regulationof apoptosis is believed to play an important role in diseasesassociated with abnormalities of cellular growth patterns such as benignprostatic hyperplasia, neurodegenerative diseases such as Parkinson'sdisease, autoimmune diseases including multiple sclerosis and rheumatoidarthritis, infectious diseases such as AIDS, and other diseases, aswell.

Compounds of this invention are also inhibitors of cGMP-specificphosphodiesterase activity found in neoplastic cells. Suchphosphodiesterases include PDE5 as well as the novel PDE disclosed inU.S. patent application Ser. No. 09/173,375 filed Oct. 15, 1998 toPamukcu et al. For convenience, the PDE inhibitory activity of suchcompounds can be tested as taught in U.S. patent application Ser. No.09/046,739 filed Mar. 24, 1998 to Pamukcu et al., which is incorporatedherein by reference. Thus, compounds of this invention are usefulinhibitors of PDE5 and may be useful in medical indications whereinhibition of that enzyme activity is desired.

As used herein, the term “precancerous lesion” includes syndromesrepresented by abnormal neoplastic, including dysplastic, changes oftissue. Examples include dysplasic growths in colonic, breast, bladderor lung tissues, or conditions such as dysplastic nevus syndrome, aprecursor to malignant melanoma of the skin. Examples also include, inaddition to dysplastic nevus syndromes, polyposis syndromes, colonicpolyps, precancerous lesions of the cervix (i.e., cervical dysplasia),esophagus, prostatic dysplasia, bronchial dysplasia, breast, bladderand/or skin and related conditions (e.g., actinic keratosis), whetherthe lesions are clinically identifiable or not.

As used herein, the term “carcinomas” refers to lesions that arecancerous. Examples include malignant melanomas, breast cancer, prostatecancer and colon cancer.

As used herein, the term “neoplasm” refers to both precancerous andcancerous lesions and hyperplasia.

As used herein, the term “halo” or “halogen” refers to chloro, bromo,fluoro and iodo groups, and the term “alkyl” refers to straight,branched or cyclic alkyl groups and to substituted aryl alkyl groups.The term “lower alkyl” refers to C₁ to C₈ alkyl groups.

The term “hydroxy-substituted lower alkyl” refers to lower alkyl groupsthat are substituted with at least one hydroxy group, preferably no morethan three hydroxy groups.

The term “—SO₂(lower alkyl)” refers to a sulfonyl group that issubstituted with a lower alkyl group.

The term “lower alkoxy” refers to alkoxy groups having from 1 to 8carbons, including straight, branched or cyclic arrangements.

The term “lower alkylmercapto” refers to a sulfide group that issubstituted with a lower alkyl group; and the term “lower alkylsulfonyl” refers to a sulfone group that is substituted with a loweralkyl group.

The term “pharmaceutically acceptable salt” refers to non-toxic acidaddition salts and alkaline earth metal salts of the compounds ofFormula I. The salts can be prepared in situ during the final isolationand purification of such compounds, or separately by reacting the freebase or acid functions with a suitable organic acid or base, forexample. Representative acid addition salts include the hydrochloride,hydrobromide, sulfate, bisulfate, acetate, valerate, oleate, palmatate,stearate, laurate, borate, benzoate, lactate, phosphate, tosylate,mesylate, citrate, maleate, fumarate, succinate, tartrate,glucoheptonate, lactobionate, lauryl sulfate salts and the like.Representative alkali and alkaline earth metal salts include the sodium,calcium, potassium and magnesium salts.

It will be appreciated that certain compounds of Formula I can possessan asymmetric carbon atom and are thus capable of existing asenantiomers. Unless otherwise specified, this invention includes suchenantiomers, including any racemates. The separate enaniomers may besynthesized from chiral starting materials, or the racemates can beresolved by conventional procedures that are well known in the art ofchemistry such as chiral chromatography, fractional crystallization ofdiastereomeric salts and the like.

Compounds of Formula I also can exist as geometrical isomers (Z and E);the Z isomer is preferred.

Compounds of this invention may be formulated into pharmaceuticalcompositions together with pharmaceutically acceptable carriers for oraladministration in solid or liquid form, or for rectal or topicaladministration, although carriers for oral administration are mostpreferred.

Pharmaceutically acceptable carriers for oral administration includecapsules, tablets, pills, powders, troches and granules. In such soliddosage forms, the carrier can comprise at least one inert diluent suchas sucrose, lactose or starch. Such carriers can also comprise, as isnormal practice, additional substances other than diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, troches and pills, the carriers may also comprise bufferingagents. Carriers such as tablets, pills and granules can be preparedwith enteric coatings on the surfaces of the tablets, pills or granules.Alternatively, the enterically coated compound can be pressed into atablet, pill, or granule, and the tablet, pill or granules foradministration to the patient. Preferred enteric coatings include thosethat dissolve or disintegrate at colonic pH such as shellac or EudragetS.

Pharmaceutically acceptable carriers include liquid dosage forms fororal administration, e.g., pharmaceutically acceptable emulsions,solutions, suspensions, syrups and elixirs containing inert diluentscommonly used in the art, such as water. Besides such inert diluents,compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, and sweetening, flavoring andperfuming agents.

Pharmaceutically acceptable carriers for topical administration includeDMSO, alcohol or propylene glycol and the like that can be employed withpatches or other liquid-retaining material to hold the medicament inplace on the skin so that the medicament will not dry out.

Pharmaceutically acceptable carriers for rectal administration arepreferably suppositories that may contain, in addition to the compoundsof this invention excipients such as cocoa butter or a suppository wax,or gel.

The pharmaceutically acceptable carrier and compounds of this inventionare formulated into unit dosage forms for administration to a patient.The dosage levels of active ingredient (i.e., compounds of thisinvention) in the unit dosage may be varied so as to obtain an amount ofactive ingredient effective to achieve lesion-eliminating activity inaccordance with the desired method of administration (i.e., oral orrectal). The selected dosage level therefore depends upon the nature ofthe active compound administered, the route of administration, thedesired duration of treatment, and other factors. If desired, the unitdosage may be such that the daily requirement for active compound is inone dose, or divided among multiple doses for administration, e.g., twoto four times per day.

The pharmaceutical compositions of this invention are preferablypackaged in a container (e.g., a box or bottle, or both) with suitableprinted material (e.g., a package insert) containing indications,directions for use, etc.

There are several general schemes for producing compounds useful in thisinvention. One general scheme (which has several sub-variations)involves the case where both R₃ and R₄ are both hydrogen. This firstscheme is described immediately below in Scheme I. The other generalscheme (which also has several sub-variations) involves the case whereat least one of R₃ and R₄ is a moiety other than hydrogen but within thescope of Formula I above. This second scheme is described below as“Scheme II.”

The general scheme for preparing compounds where both R₃ and R₄ are bothhydrogen is illustrated in Scheme I, which is described in part in U.S.Pat. No. 3,312,730, which is incorporated herein by reference. In SchemeI, R₁ is as defined in Formula I above. However, in Scheme I, thatsubstituent can also be a reactive moiety (e.g. a nitro group) thatlater can be reacted to make a large number of other substituted indenesfrom the nitro-substituted indenes.

In Scheme I, several sub-variations can be used. In one sub-variation, asubstituted benzaldehyde (a) may be condensed with a substituted aceticester in a Knoevenagel reaction (see reaction 2) or with an α-halogenopropionic ester in a Reformatsky Reaction (see reactions 1 and 3). Theresulting unsaturated ester (c) is hydrogenated and hydrolyzed to give asubstituted benzyl propionic acid (e) (see reactions 4 and 5).Alternatively, a substituted malonic ester in a typical malonic estersynthesis (see reactions 6 and 7) and hydrolysis decarboxylation of theresulting substituted ester (g) yields the benzyl propionic acid (e)directly. This latter method is especially preferable for nitro andalkylthio substituents on the benzene ring.

The next step is the ring closure of the β-aryl proponic acid (e) toform an indanone (h) which may be carried out by a Friedel-CraftsReaction using a Lewis acid catalyst (Cf. Organic Reactions, Vol. 2, p.130) or by heating with polyphosphoric acid (see reactions 8 and 9,respectively). The indanone (h) may be condensed with an α-halo ester inthe Reformatsky Reaction to introduce the aliphatic acid side chain byreplacing the carboxyl group (see reaction 10). Alternately, thisintroduction can be carried out by the use of a Wittig Reaction in whichthe reagent is a α-triphenylphosphinyl ester, a reagent which replacesthe carbonyl with a double bond to the carbon (see reaction 12). Thisproduct (1) is then immediately rearranged into the indene (j)(seereaction 13). If the Reformatsky Reaction route is used, theintermediate 3-hydroxy-3-aliphatic acid derivative i must be dehydratedto the indene (j) (see reaction 11).

The indenylacetic acid (k) in THF then is allowed to react with oxalylor thionyl chloride or similar reagent to produce the acid chloride (m)(see reaction 15), whereupon the solvent is evaporated. There are twomethods to carry out reaction 16, which is the addition of thebenzylamine side chain (n).

Method (I)

In the first method, the benzylamine (n) is added slowly at roomtemperature to a solution of 5-fluoro-2-methyl-3-indenylacetyl chloridein CH₂Cl₂. The reaction mixture is refluxed overnight, and extractedwith aqueous HCl (10%), water, and aqueous NaHCO₃ (5%). The organicphase is dried (Na₂SO₄) and is evaporated to give the amide compound (o)

Method (II)

In the second method, the indenylacetic acid (k) in DMA is allowed toreact with a carbodiimide (e.g.N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) andbenzylamine at room temperature for two days. The reaction mixture isadded dropwise to stirred ice water. A yellow precipitate is filteredoff, is washed with water, and is dried in vacuo. Recrystallizationgives the amide compound (o).

Compounds of the type a′ (Scheme III), o (Scheme I), t (Scheme II), y(Scheme IIB) may all be used in the condensation reaction shown inScheme III.

Substituents

X=halogen, usually Cl or Br.

E=methyl, ethyl or benzyl, or lower acyl.

R₁, R₂, R₆, R₅, and R₇=as defined in Formula I.

Y, n and m=as defined in Formula I.

Reagents and general conditions for Scheme I (numbers refer to thenumbered reactions):

(1) Zn dust in anhydrous inert solvent such as benzene and ether.

(2) KHSO₄ or p-toluene sulfonic acid.

(3) NaOC₂H₅ in anhydrous ethanol at room temperature.

(4) H₂ palladium on charcoal, 40 p.s.i. room temperature.

(5) NaOH in aqueous alcohol at 20-100°.

(6) NaOC₂H₅ or any other strong base such as NaH or K-t-butoxide.

(7) Acid.

(8) Friedel-Crafts Reaction using a Lewis Acid catalyst Cf. OrganicReactions, Vol. II, p. 130.

(9) Heat with polyphosphoric acid.

(10) Reformatsky Reaction: Zn in inert solvent, heat.

(11) p-Toluene sulfonic acid and CaCl₂ or I₂ at 200°

(12) Wittig Reaction using (C₆H₅)₃ P═C—COOE 20-80° in ether or benzene

(13) (a) NBS/CCl₄/benzoyl peroxide (b) PtO₂/H₂ (1 atm.)/acetic acid

(14) (a) NaOH (b) HCl

(15) Oxalyl or thionyl chloride in CH₂Cl₂ or THF

(16) Method I: 2 equivalents of NH₂—C(R₅R₆)—Ph—(R₇)_(m) Method II:carbodiimide in THF

(17) 1N NaOCH₃ in MeOH under reflux conditions

Indanones within the scope of compound (h) in Scheme I are known in theliterature and are thus readily available as intermediates for theremainder of the synthesis so that reactions 1-7 can be convenientlyavoided. Among such known indanones are:

5-methoxyindanone

6-methoxyindanone

5-methylindanone

5-methyl-6-methoxyindanone

5-methyl-7-chloroindanone

4-methoxy-7-chloroindanone

4-isopropyl-2,7-dimethylindanone

5,6,7-trichloroindanone

2-n-butylindanone

5-methylthioindanone

Scheme II has two mutually exclusive sub-schemes: Scheme IIA and SchemeII B. Scheme II A is used when R₃ is hydroxy and R₄ is hydrogen or whenthe two substituents form an oxo group. When R₃ is lower alkyl amino,Scheme II B is employed.

Similar to Scheme I, in Scheme IIA the indenylacetic acid (k) in THF isallowed to react with oxalylchloride under reflux conditions to producethe acid chloride (p) (see reaction 18), whereupon the solvent isevaporated. In reaction 19, a 0° C. mixture of a benzyl hydroxylaminehydrochloride (q) and Et₃N is treated with a cold solution of the acidchloride in CH₂Cl₂ over a period of 45-60 minutes. The mixture is warmedto room temperature and stirred for one hour, and is treated with water.The resulting organic layer is washed with 1 N HCl and brine, is driedover magnesium sulfate and is evaporated. The crude product, aN-hydroxy-N-benzyl acetamide (r) is purified by crystallization or flashchromatography. This general procedure is taught by Hoffman et al., JOC1992, 57, 5700-5707.

The next step is the preparation of the N-mesyloxy amide (s) in reaction20, which is also taught by Hoffman et al., JOC 1992, 57, 5700-5707.Specifically, to a solution of the hydroxamic acid (r) in CH₂Cl₂ at 0°C. is added triethylamine. The mixture is stirred for 10-12 minutes, andmethanesulfonyl chloride is added dropwise. The mixture is stirred at 0°C. for two hours, is allowed to warm to room temperature, and is stirredfor another two hours. The organic layer is washed with water, 1 N HCl,and brine, and is dried over magnesium sulfate. After rotaryevaporation, the product(s) is usually purified by crystallization orflash chromatography.

The preparation of the N-benzyl-α-(hydroxy) amide (t) in reaction 21, isalso taught by Hoffman et al., JOC 1992, 57, 5700-5707 and Hoffman etal., JOC 1995, 60, 4121-4125. Specifically, to a solution of theN-(mesyloxy) amide (s) in CH₃CN/H₂O is added triethylamine in CH₃CN overa period of 6-12 hours. The mixture is stirred overnight. The solvent isremoved, and the residue is dissolved in ethyl acetate. The solution iswashed with water, 1 N HCl, and brine, and is dried over magnesiumsulfate. After rotary evaporation, the product (t) is usually purifiedby recrystallization.

Reaction 22 in Scheme IIA involves a condensation with certainaldehydes, which is described in Scheme III below, a scheme that iscommon to products made in accordance with Schemes I, IIA and IIB.

The final reaction 23 in Scheme IIA is the preparation of theN-benzyl-α-ketoamide (v), which involves the oxidation of a secondaryalcohol (u) to a ketone by e.g. a Pfitzner-Moffatt oxidation, whichselectively oxidizes the alcohol without oxidizing the Y group.Compounds (u) and (v) may be derivatized in order to obtain compoundswith R₃ and R₄ groups as set forth in Formula I.

As explained above, Scheme IIB is employed when R₃ is lower alkyl amino.Similar to Scheme I, in Scheme IIB the indenylacetic acid (k) in THF isallowed to react with oxalylchloride under reflux conditions to producethe acid chloride (p) (see reaction 18), whereupon the solvent isevaporated. In reaction 24, a mixture of an alkyl hydroxylaminehydrochloride (i.e. HO—NHR where R is a lower alkyl, preferablyisopropyl) and Et₃N is treated at 0° C. with a cold solution of the acidchloride in CH₂Cl₂ over a period of 45-60 minutes. The mixture is warmedto room temperature and is stirred for one hour, and is diluted withwater. The resulting organic layer is washed with 1 N HCl and brine, isdried over magnesium sulfate and is evaporated. The crude product, aN-hydroxy-N-alkyl acetamide (w) is purified by crystallization or flashchromatography. This general procedure is also taught by Hoffman et al.,JOC 1992, 57, 5700-5707

The preparation of the N-mesyloxy amide (x) in reaction 25, which isalso taught by Hoffman et al., JOC 1992, 57, 5700-5707. Specifically, asolution of the hydroxamic acid (w) in CH₂Cl₂ at 0° C. is treated withtriethylamine, is stirred for 10-12 minutes, and is treated dropwisewith methanesulfonyl chloride. The mixture is stirred at 0° C. for twohours, is allowed to warm to room temperature, and is stirred foranother two hours. The resulting organic layer is washed with water, 1 NHCl, and brine, and is dried over magnesium sulfate. After rotaryevaporation, the product (x) is usually purified by crystallization orflash chromatography.

The preparation of the N-benzyl indenyl-α-loweralkylamino-acetamidecompound (y) in Scheme IIB as taught by Hoffman et al., JOC 1995, 60,4121-25 and J. Am. Chem Soc. 1993, 115, 5031-34, involves the reactionof the N-mesyloxy amide (x), with a benzylamine in CH₂Cl₂ at 0° C. isadded over a period of 30 minutes. The resulting solution is stirred at0° C. for one hour and at room temperature overnight. The solvent isremoved, and the residue is treated with 1 N NaOH. The extract withCH₂Cl₂ is washed with water and is dried over magnesium sulfate. Afterrotary evaporation, the product (y) is purified by flash chromatographyor crystallization.

Scheme III involves the condensation of the heterocycloaldehydes (i.e.Y—CHO) with the indenyl amides to produce the final compounds of FormulaI. This condensation is employed, for example, in reaction 17 in SchemeI above and in reaction 22 in Scheme IIA. It is also used to convertcompound (y) in Scheme IIB to final compounds of Formula I.

In Scheme III, the amide (a′) from the above schemes, aN-heterocycloaldehyde (z), and sodium methoxide (1 M in methanol) arestirred at 60° C. under nitrogen for 24 hours. After cooling, thereaction mixture is poured into ice water. A solid is filtered off, iswashed with water, and is dried in vacuo. Recrystallization provides acompound of Formula I in Schemes I and IIB and the intermediate (u) inScheme IIA.

As has been pointed out above, it is preferable in the preparation ofmany types of the compounds of this invention, to use a nitrosubstituent on the benzene ring of the indanone nucleus and convert itlater to a desired substituent since by this route a great manysubstituents can be reached. This is done by reduction of the nitro tothe amino group followed by use of the Sandmeyer Reaction to introducechlorine, bromine, cyano or xanthate in place of the amino. From thecyano derivatives hydrolysis yields the carboxamide and carboxylic acid;other derivatives of the carboxy group such as the esters can then beprepared. The xanthates, by hydrolysis, yield the mercapto group thatmay be oxidized readily to the sulfonic acid or alkylated to analkylthio group which can then be oxidized to alkylsulfonyl groups.These reactions may be carried out either before or after theintroduction of the 1-substituent.

The foregoing may be better understood from the following examples thatare presented for purposes of illustration and are not intended to limitthe scope of the invention. As used in the following examples, thereferences to substituents such as R₁, R₂, etc., refer to thecorresponding compounds and substituents in Formula I above.

EXAMPLE 1(Z)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-indenylacetamide

(A) p-Fluoro-α-Methylcinnamic Acid

p-Fluorobenzaldehyde (200 g, 1.61 mol), propionic anhydride (3.5 g, 2.42mol) and sodium propionate (155 g, 1.61 mol) are mixed in a one literthree-necked flask which had been flushed with nitrogen. The flask isheated gradually in an oil-bath to 140° C. After 20 hours, the flask iscooled to 100° C. and poured into 8 l of water. The precipitate isdissolved by adding potassium hydroxide (302 g) in 2 l of water. Theaqueous solution is extracted with ether, and the ether extracts arewashed with potassium hydroxide solution. The combined aqueous layersare filtered, are acidified with concentrated HCl, and are filtered. Thecollected solid, p-fluoro-α-methylcinnamic acid, is washed with water,and is dried and used as obtained.

(B) p-Fluoro-α-Methylhydrocinnamic Acid

To p-fluoro-α-methylcinnamic acid (177.9 g, 0.987 mol) in 3.6 l ethanolis added 11.0 g of 5% Pd/C. The mixture is reduced at room temperatureunder a hydrogen pressure of 40 p.s.i. When hydrogen uptake ceases, thecatalyst is filtered off, and the solvent is evaporated in vacuo to givethe product, p-fluoro-α-methylhydrocinnamic acid, which was useddirectly in the next step.

(C) 6-Fluoro-2-Methylindanone

To 932 g polyphosphoric acid at 70° C. (steam bath) is addedp-fluoro-α-methylhydrocinnamic acid (93.2 g, 0.5 mol) slowly withstirring. The temperature is gradually raised to 95° C., and the mixtureis kept at this temperature for 1 hour. The mixture is allowed to cooland is added to 2 l. of water. The aqueous suspension is extracted withether. The extract is washed twice with saturated sodium chloridesolution, 5% Na₂CO₃ solution, and water, and is dried, and isconcentrated on 200 g silica-gel; the slurry is added to a five poundsilica-gel column packed with 5% ether-petroleum ether. The column iseluted with 5-10% ether-petroleum ether, to give6-fluoro-2-methylindanone. Elution is followed by TLC.

(D) 5-Fluoro-2-Methylindenyl-3-Acetic Acid

A mixture of 6-fluoro-2-methylindanone (18.4 g, 0.112 mol), cyanoaceticacid (10.5 g, 0.123 mol), acetic acid (6.6 g), and ammonium acetate (1.7g) in dry toluene (15.5 ml) is refluxed with stirring for 21 hours, asthe liberated water is collected in a Dean Stark trap. The toluene isevaporated, and the residue is dissolved in 60 ml of hot ethanol and 14ml of 2.2 N aqueous potassium hydroxide solution. 22 g of 85% KOH in 150ml of water is added, and the mixture refluxed for 13 hours undernitrogen. The ethanol is removed under vacuum, and 500 ml water isadded. The aqueous solution is extracted well with ether, and is thenboiled with charcoal. The aqueous filtrate is acidified to pH 2 with 50%cold hydrochloric acid. The precipitate is dried and5-fluoro-2-methylindenyl-3-acetic acid (M.P. 164-166° C.) is obtained.

(E) 5-Fluoro-2-Methylindenyl-3-Acetyl Chloride

5-fluoro-2-methylindenyl-3-acetic acid (70 mmol) in THF (70 ml) isallowed to react with oxalylchloride (2 M in CH₂Cl₂; 35 ml; 70 mmol)under reflux conditions (24 hours). The solvent is evaporated to yieldthe title compound, which is used as such in the next step.

(F) 5-Fluoro-2-Methyl-3-(N-Benzyl)-indenylacetamide

Benzylamine (5 mmol) is added slowly at room temperature to a solutionof 5-fluoro-2-methylindenyl-3-acetyl chloride (2.5 mmol.) in CH₂Cl₂ (10ml). The reaction mixture is refluxed overnight, and is extracted withaqueous HCl (10%), water, and aqueous NaHCO₃ (5%). The organic phase isdried (Na₂SO₄) and is evaporated to give the title compound, which isrecrystallized from CH₂Cl₂ to give the title compound as a white solid(m.p. 144° C.).

(G)(Z)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-indenylacetamide

5-fluoro-2-methyl-3-(N-benzyl)-indenylacetamide (3.38 mmol),4-pyridinecarboxaldehyde (4 mmol), sodium methoxide (1M NaOCH₃ inmethanol (30 ml)) are heated at 60° C. under nitrogen with stirring for24 hours. After cooling, the reaction mixture is poured into ice water(200 ml). A solid is filtered off, washed with water, and dried invacuo. Recrystallization from CH₃CN gives the title compound (m.p. 202°C.) as a yellow solid (R₁═F, R₂═CH₃, R₃═H, R₄═H, R₅═H, R₆═H, R₆═H,RE₇═H, n=1, m=1, Y=4-pyridinyl).

(H)(E)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-indenylacetamide

The mother liquor obtained from the CH₃CN recrystallization of 1G isrich on the geometrical isomer of 1G. The E-isomer can be obtained pureby repeated recrystallizations from CH₃CN.

EXAMPLE 2(Z)-5-Fluoro-2-Methyl-(3-Pyridinylidene)-3-(N-Benzyl)-indenylacetamide

This compound is obtained from5-fluoro-2-methyl-3-(N-benzyl)-indenylacetamide (Example 1F) using theprocedure of Example 1, part G and replacing 4-pyridinecarboxaldehydewith 3-pyridinecarboxaldehyde. Recrystallization from CH₃CN gives thetitle compound (m.p. 175° C.)(R₁═F, R₂═CH₃, R₃═H, R₄═H, R₅═H, R₆═H,R₇═H, n=1, m=1, Y=3-pyridinyl).

EXAMPLE 3(Z)-5-Fluoro-2-Methyl-(2-Pyridinylidene)-3-(N-Benzyl)-indenylacetamide

This compound is obtained from5-fluoro-2-methyl-3-(N-benzyl)-indenylacetamide (Example 1F) using theprocedure of Example 1, part G and replacing 4-pyridinecarboxaldehydewith 2-pyridinecarboxaldehyde. Recrystallization from ethylacetate givesthe title compound (m.p. 218° C.)(R₁═F, R₂═CH₃, R₃═H, R₄═H, R₅═H, R₆═H,R₇═H, n=1, m=1, Y=2-pyridinyl).

EXAMPLE 4(Z)-5-Fluoro-2-Methyl-(4-Quinolinylidene)-3-(N-Benzyl)-indenylacetamide

This compound is obtained from5-fluoro-2-methyl-3-(N-benzyl)-indenylacetamide (Example 1F) using theprocedure of Example 1, part G and replacing 4-pyridinecarboxaldehydewith 4-quinolinecarboxaldehyde. Recrystallization from ethylacetategives the title compound (m.p. 239° C.)(R₁═F, R₂═CH₃, R₃═H, R₄═H, R₅═H,R₆═H, R₇═H, n=1, m=1, Y=4-quinolinyl).

EXAMPLE 5(Z)-5-Fluoro-2-Methyl-(4,6-Dimethyl-2-Pyridinylidene)-3-(N-Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 4,6-dimethyl-2-pyridinecarboxaldehyde accordingto the procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁═F, R₂═CH₃,R₃═H, R₄═H, R₅═H, R₆═H, R₇═H, n=1, m=1, Y=4,6-dimethyl-2-pyridinyl).

EXAMPLE 6(Z)-5-Fluoro-2-Methyl-(3-Quinolinylidene)-3-(N-Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 3-quinolinecarboxaldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁═F, R₂═CH₃, R₃═H, R₄═H,R₅═H, R₆H, R₇═H, n=1, m=1, Y=3-quinolinyl)

EXAMPLE 7(Z)-5-Fluoro-2-Methyl-(2-Quinolinylidene)-3-(N-Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 2-quinolinecarboxaldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁═F, R₂═CH₃, R₃═H, R₄═H,R₅═H, R₆═H, R₇═H, n=1, m=1, Y=2-quinolinyl).

EXAMPLE 8(Z)-5-Fluoro-2-Methyl-(Pyrazinylidene)-3-(N-Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with pyrazinealdehyde according to the procedure ofExample 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁═F, R₂═CH₃, R₃═H, R₄═H,R₅═H, R₆═H, R₇═H, n=1, m=1, Y=pyrazinyl).

EXAMPLE 9(Z)-5-Fluoro-2-Methyl-(3-Pyridazinylidene)-3-(N-Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with pyridazine-3-aldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁═F, R₂═CH₃, R₃═H, R₄═H,R₅═H, R₆═H, R₇═H, n=1, m=1, Y=3-pyridazinyl).

EXAMPLE 10(Z)-5-Fluoro-2-Methyl-(4-Pyrimidinylidene)-3-(N-Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with pyrimidine-4-aldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁═F, R₂═CH₃, R₃═H, R₄═H,R₅═H, R₆═H, R₇═H, n=1, m=1, Y=4-pyrimidinyl).

EXAMPLE 11(Z)-5-Fluoro-2-Methyl-(2-Methyl-4-Pyrimidinylidene)-3-(N-Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 2-methyl-pyrimidine-4-aldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁═F, R₂═CH₃, R₃═H, R₄═H,R₅═H, R₆═H, R₇═H, n=1, m=1, Y=2-methyl-4-pyrimidinyl).

EXAMPLE 12(Z)-5-Fluoro-2-Methyl-(4-Pyridazinylidene)-3-(N-Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with pyridazine-4-aldehyde according to theprocedure of Example 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁═F, R₂═CH₃, R₃═H, R₄═H,R₅═H, R₆═H, R₇═H, n=1, m=1, Y=4-pyridazinyl).

EXAMPLE 13(Z)-5-Fluoro-2-Methyl-(1-Methyl-3-Indolylidene)-3-(N-Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 1-methylindole-3-carboxaldehyde according tothe procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁═F, R₂═CH₃,R₃═H, R₄═H, R₅═H, R₆═H, R₇═H, n=1, m=1, Y=1-methyl-3-indolyl).

EXAMPLE 14(Z)-5-Fluoro-2-Methyl-(1-Acetyl-3-Indolylidene)-3-(N-Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-benzyl)-indenylacetamide from Example 1, part Fis allowed to react with 1-acetyl-3-indolecarboxaldehyde according tothe procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁═F, R₂═CH₃,R₃═H, R₄═H, R₅═H, R₆═H, R₇═H, n=1, m=1, Y=1-acetyl-3-indolyl).

EXAMPLE 15 (Z)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3(N-2-Fluorobenzyl)-indenylacetamide

(A) 5-Fluoro-2-Methyl-3-(N-2-Fluorobenzyl)-indenylacetamide

This compound is obtained from 5-fluoro-2-methylindenyl-3-acetylchloride (Example 1E) using the procedure of Example 1, Part F andreplacing benzylamine with 2-fluorobenzylamine.

(B)(Z)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3-(N-2-Fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide is allowed toreact with 4-pryidinecarboxaldehyde according to the procedure ofExample 1, part G in order to obtain the title compound.Recrystallization gives the title compound (R₁═F, R₂═CH₃, R₃═H, R₄═H,R₅═H, R₆═H, R₇═F, n=1, m=1, Y=4-pyridinyl).

EXAMPLE 16(Z)-5-Fluoro-2-Methyl-(3-Pyridinylidene)-3-(N-2-Fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with 3-pryidinecarboxaldehyde according tothe procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁═F, R₂═CH₃,R₃═H, R₄═H, R₅═H, R₆═H, R₇═F, n=1, m=1, Y=3-pyridinyl).

EXAMPLE 17(Z)-5-Fluoro-2-Methyl-(2-Pyridinylidene)-3-(N-2-Fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with 2-pyridinecarboxaldehyde according tothe procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁═F, R₂═CH₃,R₃═H, R₄═H, R₅═H, R₆═H, R₇═F, n=1, m=1, Y=2-pyridinyl).

EXAMPLE 18(Z)-5-Fluoro-2-Methyl-(4-Quinolinylidene)-3-(N-2-Fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with 4-quinolinecarboxaldehyde according tothe procedure of Example 1, part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁═F, R₂═CH₃,R₃═H, R₄═H, R₅═H, R₆═H, R₇═F, n=1, m=1, Y=3-quinolinyl).

EXAMPLE 19(Z)-5-Fluoro-2-Methyl-(3-Pyrazinylidene)-3-(N-2-Fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with pyrazinealdehyde according to theprocedure of Example 1, Part G in order to obtain the title compound.Recrystallization gives the title compound (R₁═F, R₂═CH₃, R₃═H, R₄═H,R₅═H, R₆═H, R₇═F, n=1, m=1, Y=3-pyrazinyl).

EXAMPLE 20(Z)-5-Fluoro-2-Methyl-(3-Pyridazinylidene)-3-(N-2-Fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with 3-pryidaziine-3-aldehyde according tothe procedure of Example 1, Part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁═F, R₂═CH₃,R₃═H, R₄═H, R₅═H, R₆═H, R₇═F, n=1, m=1, Y=3-pyridazinyl).

EXAMPLE 21(Z)-5-Fluoro-2-Methyl-(3-Pyrimidinylidene)-3-(N-2-Fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with pryimidine-4-aldehyde according to theprocedure of Example 1, Part G in order to obtain the title compound.Recrystallization gives the title compound (R₁═F, R₂═CH₃, R₃═H, R₄═H,R₅═H, R₆═H, R₇═F, n=1, m=1, Y=3-pyrimidinyl).

EXAMPLE 22(Z)-5-Fluoro-2-Methyl-(4-Pyridazinylidene)-3-(N-2-Fluorobenzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-2-fluorobenzyl)-indenylacetamide from Example 15,part A is allowed to react with pryidazine-4-aldehyde according to theprocedure of Example 1, Part G in order to obtain the title compound.Recrystallization gives the title compound (R₁═F, R₂═CH₃, R₃═H, R₄═H,R₅═H, R₆═H, R₇═F, n=1, m=1, Y=4-pyridazinyl).

EXAMPLE 23(Z)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-indenylacetamide

(A) 5-Fluoro-2-Methyl-3-(N-(S-α-Hydroxylmethyl)Benzyl)-indenylacetamide

5-Fluoro-2-methylindenyl-3-acetic acid (from Example 1D) (2.6 mmol) inDMA (2 ml) is allowed to react withn-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (4 mmol)and S-2-amino-2-phenylethanol (3.5 mmol) at room temperature for twodays. The reaction mixture is added dropwise to stirred ice water (50ml). A white precipitate is filtered off, washed with water (5 ml), anddried in vacuo. Recrystallization from ethylacetate gives the desiredcompound.

(B)(Z)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide frompart A is allowed to react with 4-pryidinecarboxaldehyde according tothe procedure of Example 1, Part G in order to obtain the titlecompound. Recrystallization gives the title compound (R₁═F, R₂═CH₃,R₃═H, R₄═H, R₅═CH₂OH, R₆═H, R₇═H, n=1, m=1, Y=4-pyridinyl).

EXAMPLE 24(Z)-5-Fluoro-2-Methyl-(3-Pyridinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with 3-pryidinecarboxaldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁═F,R₂═CH₃, R₃═H, R₄═H, R₅═CH₂OH, R₆═H, R₇═H, n=1, m=1, Y=3-pyridinyl).

EXAMPLE 25(Z)-5-Fluoro-2-Methyl-(2-Pyridinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with 2-pryidinecarboxaldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁═F,R₂═CH₃, R₃═H, R₄═H, R₅═CH₂OH, R₆═H, R₇═H, n=1, m=1, Y=2-pyridinyl).

EXAMPLE 26(Z)-5-Fluoro-2-Methyl-(4-Quinolinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with 4-quinolinecarboxaldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁═F,R₂═CH₃, R₃═H, R₄═H, R₅═CH₂OH, R₆═H, R₇═H, n=1, m=1, Y=4-quinolinyl).

EXAMPLE 27(Z)-5-Fluoro-2-Methyl-(Pyrazidinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with pryazidinecarboxaldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁═F,R₂═CH₃, R₃═H, R₄═H, R₅═CH₂OH, R₆═H, R₇═H, n=1, m=1, Y=pyrazidinyl).

EXAMPLE 28(Z)-5-Fluoro-2-Methyl-(3-Pyridazinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with pryidazine-3-aldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁═F,R₂═CH₃, R₃═H, R₄═H, R₅═CH₂OH, R₆═H, R₇═H, n=1, m=1, Y=3-pyridazinyl).

EXAMPLE 29(Z)-5-Fluoro-2-Methyl-(4-Pyrimidinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with pryimidine-4-aldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁═F,R₂═CH₃, R₃═H, R₄═H, R₅═CH₂OH, R₆═H, R₇═H, n=1, m=1, Y=4-pyrimidinyl).

EXAMPLE 30(Z)-5-Fluoro-2-Methyl-(4-Pyridazinylidene)-3-(N-(S-α-Hydroxymethyl)Benzyl)-indenylacetamide

5-Fluoro-2-methyl-3-(N-(S-α-hydroxylmethyl)benzyl)-indenylacetamide fromExample 23 part A is allowed to react with pryidazine-4-aldehydeaccording to the procedure of Example 1, Part G in order to obtain thetitle compound. Recrystallization gives the title compound (R₁═F,R₂═CH₃, R₃═H, R₄═H, R₅═CH₂OH, R₆═H, R₇═H, n=1, m=1, Y=4-pyridazinyl).

EXAMPLE 31rac-(Z)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)Indenyl-α-Hydroxyacetamide

(A) 5-Fluoro-2-Methyl-3-(N-Benzyl-N-Hydroxy)-Indenylacetamide

To a mixture of N-benzylhydroxylamine hydrochoride (12 mmol) and Et₃N(22 mmol) in CH₂Cl₂ (100 ml) at 0° C. is added a cold solution of5-fluoro-2-methylindenyl-3-acetyl chloride (Example 1, Step E) (10 mmol)in CH₂Cl₂ (75 ml) over a period of 45-60 minutes. The mixture is warmedto room temperature and is stirred for 1 hour. The mixture is dilutedwith water (100 ml), and the organic layer is washed with HCl (2×25 ml)and brine (2×100 ml), dried (MgSO₄) and evaporated. The crude product ispurified with flash chromatography to give the title compound.

(B) 5-Fluoro-2-Methyl-3-(N-Benzyl-N-Mesyloxy)-Indenylacetamide

To a solution of5-fluoro-2-methyl-3-(N-benzyl-N-hydroxy)-indenylacetamide (5 mmol) inCH₂Cl₂ (25 ml) at 0° C. is added triethylamine (5 mmol). The mixture isstirred for 10 minutes, and methanesulfonyl chloride (5.5 mmol) is addeddropwise. The solution is stirred at 0° C. for 2 hours, allowed to warmto room temperature, and stirred for another 2 hours. The organic layeris washed with water (2×20 ml), in HCl (15 ml), and brine (20 ml) anddried over MgSO₄. After rotary evaporation, the product is purified withflash chromatography to give the title compound.

(C) Rac-5-Fluoro-2-Methyl-3-(N-Benzyl)-α-Hydroxyindenylacetamide

To a solution of5-fluoro-2-methyl-3-(N-benzyl-N-mesyloxy)-indenylacetamide (2 mmol) inCH₃CN/H₂O (12 ml. each) is added triethylamine (2.1 mmol) in CH₃CN (24ml) over a period of 6 hours. The mixture is stirred overnight. Thesolvent is removed, and the residue diluted with ethyl acetate (60 ml),washed with water (4×20 ml), in HCl (15 ml), and brine (20 ml) and driedover MgSO₄. After rotary evaporation, the product is purified byrecrystallization to give the title compound.

(D)rac-(Z)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenyl-α-hydroxyacetamideis obtained fromrac-5-fluoro-2-methyl-3-(N-benzyl)-(α-hydroxyindenylacetamide using theprocedure of Example 1, Part G (R₁═F, R₂═CH₃, R₃═OH, R₄═H, R₅═H, R₆═H,R₇═H, n=1, m=1, Y=4-pyridinyl).

EXAMPLE 322-[(Z)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-indenyl]-Oxyacetamide

For Pfitzner-Moffatt oxidation, a solution ofrac-(Z)-5-fluoro-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenyl-α-hydroxyacetamide(1 mmol) in DMSO (5 ml) is treated with dicyclohexylcarbodiimide (3mmol). The mixture is stirred overnight, and the solvent is evaporated.The crude product is purified by flash chromatography to give the titlecompound (R₁═F, R₂═CH₃, R₃ and R₄ together form C═O, R₅═H, R₆═H, R₇═H,n=1, m=1, and Y=4-pyridinyl).

EXAMPLE 33rac-(Z)-5-Fluoro-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-indenyl-(α-(2-Propylamino)-Acetamide

(A) 5-Fluoro-2-methyl-3-(N-2-propyl-N-hydroxy)-indenylacetamide isobtained from 5-fluoro-2-methylindenyl-3-acetyl chloride (Example 1,Step E) using the procedure of Example 31, Part A and replacingN-benzylhydroxylamine hydrochloride with N-2-propyl hydroxylaminehydrochloride.

(B) 5-Fluoro-2-methyl-3-(N-2-propyl-N-mesyloxy)-indenylacetamide isobtained according to the procedure of Example 31, Part B.

(C) rac-5-Fluoro-2-methyl-3-(N-benzyl)-α-(2-propylamino)-acetamide. To5-fluoro-2-methyl-3-(N-2-propyl-N-mesyloxy)-indenylacetamide (2 mmol) inCH₂Cl₂ (25 ml) at 0° C. is added benzylamine (4.4 mmol) in CH₂Cl₂ (15ml) over a period of 30 minutes. The resulting solution is stirred at 0°C. for 1 hour, and at room temperature overnight. The solvent isremoved, and the residue is treated with 1 N NaOH, and is extracted withCH₂Cl₂ (100 ml). The extract is washed with water (2×10 ml), and isdried over MgSO₄. After rotary evaporation, the product is purified byflash chromatography.

(D)rac-(Z)-5-Fluoro-2-methyl-(4-pyridinylidene)-3-(N-benzyl)-indenyl-α-(2-propylamino)-acetamideis obtained fromrac-5-fluoro-2-methyl-3-(N-benzyl)-α-(2-propylamino)-acetamide using theprocedure of Example 1, Part G (R₁═F, R₂═CH₃, R₃=isopropylamino, R₄═H,R₅═H, R₆═H, R₇═H, n=1, m=1, Y=4-pyridinyl).

EXAMPLE 34(Z)-6-Methoxy-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-indenylacetamide

(A) Ethyl-2-Hydroxy-2-(p-Methoxyphenyl)-1-Methylpropionate

In a 500 ml. 3-necked flask is placed 36.2 g. (0.55 mole) of zinc dust,a 250 ml. addition funnel is charged with a solution of 80 ml. anhydrousbenzene, 20 ml. of anhydrous ether, 80 g. (0.58 mole) of p-anisaldehydeand 98 g. (0.55 mole) of ethyl-2-bromoproplonate. About 10 ml. of thesolution is added to the zinc dust with vigorous stirring, and themixture is warmed gently until an exothermic reaction commences. Theremainder is added dropwise at such a rate that the reaction mixturecontinues to reflux smoothly (ca. 30-35 min.). After addition iscompleted the mixture is placed in a water bath and refluxed for 30minutes. After cooling to 0°, 250 ml. of 10% sulfuric acid is added withvigorous stirring. The benzene layer is extracted twice with 50 ml.portions of 5% sulfuric acid and washed twice with 50 ml. portions ofwater. The combined aqueous acidic layers are extracted with 2×50 ml.ether. The combined etheral and benzene extracts are dried over sodiumsulfate. Evaporation of solvent and fractionation of the residue througha 6″ Vigreux column affords 89 g. (60%) of the product,ethyl-2-hydroxy-2-(p-methoxyphenyl)-1-methylpropionate, B.P. 165-160°(1.5 mm.).

(B) 6-Methoxy-2-Methylindanone

By the method described in Vander Zanden, Rec. Trav. Chim., 68, 413(1949), the compound from part A is converted to6-methoxy-2-methylindanone.

Alternatively, the same compound can be obtained by addingα-methyl-β-(p-methoxylphenyl)propionic acid (15 g.) to 170 g. ofpolyphosphoric acid at 50° and heating the mixture at 83-90° for twohours. The syrup is poured into iced water. The mixture is stirred forone-half hour, and is extracted with ether (3×). The etheral solution iswashed with water (2×) and 5% NaHCO₃ (5×) until all acidic material hasbeen removed, and is dried over sodium sulfate. Evaporation of thesolution gives 9.1 g. of the indanone as a pale yellow oil.

(C)(Z)-6-Methoxy-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-indenylacetamide

In accordance with the procedures described in Example 1, parts D-G,this compound is obtained substituting 6-methoxy-2-methylindanone for6-fluoro-2-methylindanone in part D of Example 1.

EXAMPLE 35(Z)-5-Methoxy-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-indenylacetamide

(A) Ethyl 5-Methoxy-2-Methyl-3-Indenyl Acetate

A solution of 13.4 g of 6-methoxy-2-methylindanone and 21 g. of ethylbromoacetate in 45 ml. benzene is added over a period of five minutes to21 g. of zinc amalgam (prepared according to Org. Syn. Coll. Vol. 3) in110 ml. benzene and 40 ml. dry ether. A few cyrstals of iodine are addedto start the reaction, and the reaction mixture is maintained at refluxtemperature (ca. 65°) with external heating. At three-hour intervals,two batches of 10 g. zinc amalgam and 10 g. bromoester are added and themixture is then refluxed for 8 hours. After addition of 30 ml. ofethanol and 150 ml. of acetic acid, the mixture is poured into 700 ml.of 50% aqueous acetic acid. The organic layer is separated, and theaqueous layer is extracted twice with ether. The combined organic layersare washed thoroughly with water, ammonium hydroxide and water. Dryingover sodium sulfate, evaporation of solvent in vacuo followed by pumpingat 80° (bath temperature)(1-2 mm.) gives crudeethyl-(1-hydroxy-2-methyl-6-methoxy-indenyl) acetate (ca. 18 g.).

A mixture of the above crude hydroxyester, 20 g. of p-toluenesulfonicacid monohydrate and 20 g. of anhydrous calcium chloride in 250 ml.toluene is refluxed overnight. The solution is filtered, and the solidresidue is washed with toluene. The combined toluene solution is washedwith water, sodium bicarbonate, water and then dried over sodiumsulfate. After evaporation, the crude ethyl 5-methoxy-2-methyl-3-indenylacetate is chromatographed on acid-washed alumina and the product iseluted with petroleum ether-ether (v./v. 50-100%) as a yellow oil (11.8g., 70%).

(B)(Z)-5-Methoxy-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-indenylacetamide

In accordance with the procedures described in Example 1, parts E-G,this compound is obtained substitutingethyl-5-methoxy-2-methyl-3-indenyl acetate for5-fluoro-2-methindenyl-3-acetic acid in Example 1, part E.

EXAMPLE 36(Z)-α-5-Methoxy-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)-indenylpropionamide

(A) α-(5-Methoxy-2-Methyl-3-Indenyl)Propionic Acid

The procedure of Example 35, part (A) is followed using ethylα-bromopropionate in equivalent quantities in place of ethylbromoacetate used therein. There is obtained ethylα-(1-hydroxy-6-methoxy-2-methyl-1-indanyl)propionate, which isdehydrated to ethyl α-(5-methoxy-2-methyl-3-indenyl)propionate in thesame manner.

The above ester is saponified to giveα-(5-methoxy-2-methyl-3-indenyl)propionic acid.

(B) (Z)-α-5-Methoxy-2-Methyl-(4-Pyridinyl)-3-(N-Benzyl)-α-Methylindenylpropionamide

In accordance with the procedures described in Example 1, parts E-G,this compound is obtained substitutingα-5-methoxy-2-methyl-3-indenyl)propionic acid for5-fluoro-2-methylindenyl-3-acetic acid in Example 1, part E.

EXAMPLE 37 (Z)α-Fluoro-5-Methoxy-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)Indenylacetamide

(A) Methyl-5-Methoxy-2-Methyl-3-Indenyl-α-Fluoro Acetate

A mixture of potassium fluoride (0.1 mole) andmethyl-5-methoxy-2-methyl-3-indenyl-α-tosyloxy acetate (0.05 mole) in200 ml. dimethylformamide is heated under nitrogen at the refluxtemperature for 2-4 hours. The reaction mixture is cooled, poured intoiced water and then extracted with ether. The ethereal solution iswashed with water, sodium bicarbonate and dried over sodium sulfate.Evaporation of the solvent and chromatography of the residue on anacid-washed alumina column (300 g.) using ether-petroleum ether (v./v.20-50%) as eluent give the product,methyl-5-methoxy-2-methyl-3-indenyl-α-fluoroacetate.

(B) (Z)α-Fluoro-5-Methoxy-2-Methyl-(4-Pyridinylidene)-3-(N-Benzyl)Indenylacetamide

In accordance with the procedures described in Example 1, parts E-G,this compound is obtained substitutingmethyl-5-methoxy-2-methyl-3-indenyl-α-fluoroacetate for5-fluoro-2-methylindenyl-3-acetic acid in Example 1, part E.

For the introduction of the ═CH—Y part in Scheme III, any of theappropriate heterocyclic aldehydes may be used either directly in thebase-catalyzed condensation or in a Wittig reaction in an alternativeroute. The aldehydes that may be used are listed in Table 1 below:

TABLE 1 pyrrol-2-aldehyde* pyrimidine-2-aldehyde6-methylpyridine-2-aldehyde* 1-methylbenzimidazole-2-aldehydeisoquinoline-4-aldehyde 4-pyridinecarboxaldehyde*3-pyridinecarboxaldehyde* 2-pyridinecarboxaldehyde*4,6-dimethyl-2-pyridinecarboxaldehyde* 4-methyl-pyridinecarboxaldehyde*4-quinolinecarboxaldehyde* 3-quinolinecarboxaldehyde*2-quinolinecarboxaldehyde* 2-chloro-3-quinolinecarboxaldehyde*pyrazinealdehyde (Prepared as described by Rutner et al., JOC 1963, 28,1898-99) pyridazine-3-aldehyde (Prepared as described by Heinisch etal., Monatshefte Fuer Chemie 108 213-224, 1977) pyrimidine-4-aldehyde(Prepared as described by Bredereck et al., Chem. Ber. 1964, 97 3407-17)2-methyl-pyrimidine-4-aldehyde (Prepared as described by Bredereck etal., Chem. Ber. 1964, 97 3407-17) pyridazine-4-aldehyde (Prepared asdescribed by Heinisch et al., Monatshefte Fuer Chemie 104 1372-1382(1973)) 1-methylindole-3-carboxaldehyde*1-acetyl-3-indolecarboxaldehyde* *Available from Aldrich

The aldehydes above can be used in the reaction schemes above incombination with various appropriate amines to produce compounds withthe scope of this invention. Examples of appropriate amines are thoselisted in Table 2 below:

TABLE 2 benzylamine 2,4-dimethoxybenzylamine 2-methoxybenzylamine2-fluorobenzylamine 4-dimethylaminobenzylamine 4-sulfonaminobenzylamine1-phenylethylamine (R-enantiomer) 2-amino-2-phenylethanol (S-enantiomer)2-phenylglycinonitrile (S-enantiomer)

EXAMPLE 38 (Z)-5-Fluoro-2-Methyl-(4-Pyridylidene)-3-(N-Benzyl)Indenylacetamide Hydrochloride

(Z)-5-Fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamide(1396 g; MW 384.45; 3.63 mol) from Example 1 is dissolved at 45° C. inethanol (28 L). Aqueous HCl (12 M; 363 mL) is added stepwise. Thereaction mixture is heated under reflux for 1 hour, is allowed to coolto room temperature, then stored at −10° C. for 3 hours. The resultingsolid is filtered off, is washed with ether (2×1.5 L) and is air-driedovernight. Drying under vacuum at 70° C. for 3 days gives(Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride with a melting point of 207-209° C. (R₁═F, R₂═CH₃, R₃═H,R₄═H, R₅═H, R₆═H, R₇═H, n=1, m=1, Y=4-pyridinyl·hydrochloride). Yield:1481 g (97%; 3.51 mol); MW: 420.91 g/mol.

¹H-NMR (DMSO-d₆): 2.18 (s,3,═C—CH₃); 3.54 (s,2,=CH₂CO); 4.28 (d,2,NCH₂); 6.71 (m,1,ar.); 7.17 (m,8,ar.); 8.11 (d,2,ar., AB system); 8.85(m,1,NH); 8.95 (d,2,ar.,AB system); IR (KBr): 3432 NH; 1635 C═O; 1598C═C.

Biological Effects

(A) Growth Inhibition

The compound of Example 1 was assayed for its growth inhibitory activityon the human colon carcinoma cell line, SW-480 obtained from ATCC(Rockville, Md.), to ascertain the degree of growth inhibition. Growthinhibition of this cell line is indicative of a benefit on precancerouslesions and neoplasms. The cell line and growth assay employed for suchexperiments are well characterized, and are used to evaluate theanti-neoplastic properties of NSAIDs. The assay is used by the UnitedStates National Cancer Institute in its screening program for newanti-cancer drugs.

Drug stock solutions were made in 100% DMSO and were then diluted withRPMI media for cell culture testing. All drug solutions were preparedfresh on the day of testing. The cultured cells were obtained at passage#99 and grown in RPMI media supplemented with 5% fetal calf serum, and 2mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 0.25 μg/mlamphotericin. The cultures were maintained in a humidified atmosphere of95% air and 5% CO₂ at 37° C. The cultures were passaged at preconfluentdensities using a solution of 0.05% trypsin and 0.53 mM EDTA. Cells wereplated at 1000 cells/well for 96 well flat-bottom microtiter plates.

Tumor cell growth inhibition was assessed using the Sulforhodamine B(SRB) protein binding assay. In this assay, tumor cells were plated in96-well plates and treated with drug-containing media for six days(continuous exposure). For each plate, 6 wells were designated as notreatment controls, six wells as vehicle (0.1% DMSO) controls, and theremaining wells for drug dilutions with three wells per drugconcentration. At the end of the exposure period, the cells were fixedand stained with sulforhodamine B, a protein binding dye. The dye wasthen solubilized, and the optical density of the resulting solution wasdetermined on a 96-well plate reader. The mean dye intensity of thetreated wells was then divided by the mean dye intensity in the controlwells (6 wells of each) to determine the effect of the drug on thecells. Dye intensity is proportional to the number of cells or amount ofprotein per well. The resultant “percent inhibition” value thenrepresents the degree of growth inhibition caused by the drug.

For each experiment, an IC₅₀ value was determined and used forcomparative purposes. This value is equivalent to the concentration ofdrug needed to inhibit tumor cell growth by 50%. IC₅₀ value was obtainedgraphically by connecting the mean values for each drug concentrationtested. Each experiment included at least three wells per drugconcentration. Concentration was plotted on a log scale on the X-axis.IC₅₀ value obtained for the compound of Example 1 wa 0.724 for theSW-480 cell line.

(B) Cyclooxygenase (COX) Inhibition

COX catalyzes the formation of prostaglandins and thromboxane by theoxidative metabolism of arachidonic acid. The compound of Example 1 ofthis invention, as well as a positive control, (sulindac sulfide) wereevaluated to determine whether they inhibited purified cyclooxygenaseType I (see Table 3 below).

The compounds of this invention were evaluated for inhibitory effects onpurified COX. The COX was purified from ram seminal vesicles, asdescribed by Boopathy, R. and Balasubramanian, J., 239:371-377, 1988.COX activity was assayed as described by Evans, A. T., et al., “Actionsof Cannabis Constituents on Enzymes Of Arachidonate MetabolismAnti-Inflammatory Potential,” Biochem. Pharmacol., 36:2035-2037, 1987.Briefly, purified COX was incubated with arachidonic acid (100 μM) for2.0 min at 37° C. in the presence or absence of test compounds. Theassay was terminated by the addition of TCA, and COX activity wasdetermined by absorbance at 530 nm.

TABLE 3 COX I EXAMPLE % Inhibition (100 μM) (* − 1000 · M) Sulindacsulfide   86 1 <25 (C) Apoptosis

Apoptosis was measured using an assay of cell death based onmorphological characteristics of apoptotic cells (i.e., condensedchromatin). Drug preparation and cell culture conditions were the sameas for the SRB assay described above, except that HT-29 human coloncarcinoma cells were used. Confluent cultures were established in 12.5cm² flasks by plating 0.5×10⁶ cells/flask. The cultures were assayed forapoptosis by fluorescent microscopy following labeling with acridineorange and ethidium bromide. Floating and attached cells were collectedby trypsinization and washed three times in PBS. One ml aliquots werecentrifuged (3 g). The pellet was resuspended in 25 μl media and 1 μl ofa dye mixture containing 100 μg/ml acridine orange and 100 μg/mlethidium bromide prepared in PBS and mixed gently. Ten μl of the mixturewas placed on a microscope slide and covered with a 22 mm² coverslip,was examined with 40× dry objectives under epillumination by filtercombination.

An observer blinded in regard to the identity of the samples scored atleast 100 cells per sample. Apoptotic cells were identified by nuclearcondensation of chromatin stained by the acridine orange or ethidiumbromide. These results are provided in Table 4 below.

TABLE 4 Apoptosis Effects of Compounds Morphology DNA Fragmentation %Apoptotic Cells FS EC₅₀ EXAMPLE (1 μM) (100 μM) (μM) 1 88 4.2 29 2 5.4 38.5 4 3.9 38 15

Apoptosis was also measured based on the amount of fragmented DNAcontained in cell lysates. Briefly, SW-480 colon adenocarcinoma cellswere plated in 96-well microtitre plates (“MTP”) at a density of 10Kcells/well in 180 μl and were incubated for 24 hrs. Cells were thentreated with 20 μl aliquots of appropriately diluted compound, andallowed to incubate for an additional 48 hrs.

After the incubation, samples were prepared according to the followingsteps. The MTP was centrifuged (15 min., 1000 rpm) and the supernatantwas carefully removed by fast inversion of the MTP. The cell pellets ineach well were resuspended in 2001 μl lysis buffer and incubated for 45min. at room temperature to lyse the cells. The lysates were thencentrifuged (15 min., 1000 rpm) and 20 μl aliquots of the supernatant(=cytoplasmic fraction) were transferred into the streptavidin coatedMTP for analysis. Care was taken not to shake the lysed pellets in theMTP (=cell nucleii containing high molecular weight, unfragmented DNA).Samples were analyzed immediately, because storage at 4C or −20C reducesthe ELISA-signals.

Samples were then processed according to a DNA fragmentation assayprotocol, and dose-response curves were generated based on opticaldensity readings. Quantification of DNA was done by a commerciallyavailable photometric enzyme-immunoassay manufactured byMannheim-Boehringer under the name “Cell Death Detection ELISA^(plus)”.The assay is based on a quantitativesandwich-enzyme-immunoassay-principle using mouse monoclonal antibodiesdirected against DNA and histones, respectively. This allows thespecific determination of mono and oligonucleosomes in the cytoplasmaticfraction of cell lysates. In brief, the assay procedure is as follows.The sample (cell-lysate, serum, culture-supernatant etc.) is placed intoa streptavidin-coated MTP. Subsequently, a mixture ofanti-histone-biotin and anti-DNA-POD is followed by incubation for 2hours. During the incubation period, the anti-histone antibody binds tothe bistone-component of the nucleosomes and simultaneously fixes theimmunocomplex to the streptavidin-coated MTP via its biotinylation.Additionally, the anti-DNA-POD antibody reacts with the DNA component ofthe nucleosomes. After removal of unbound antibodies by a washing step,the amount of nucleosomes is quantified by the POD retained in theimmunocomplex. POD is determined photometrically with ABTS®(2,2′-Azino-di[3-ethylbenzthiazolin-sulfonat])* as substrate.

Fold stimulation (FS=ODmax/ODveh), an indicator of apoptotic response,was determined for each compound tested. EC₅₀ values were determinedeither specifically by data analysis software, or by estimates based onthe effective concentration range of each compound (ECR=min. effectivedose-min. dose to peak effect). These FS and EC₅₀ values for the testedcompounds are listed above in Table 4.

In addition, using the DNA fragmentation test above, a dose response forthe compound of Example 1 was obtained. Those data are set forth inTable 5.

TABLE 5 Apoptosis Level Dose (μM) (Mean OD Value ± SD) 0.5 0.186 ± 0.0081.0 0.207 ± 0.061 5.0 0.208 ± 0.073 10 0.296 ± 0.050 50 0.500 ± 0.048100 0.633 ± 0.053 500 0.659 ± 0.012

The compounds of this invention can be formulated with pharmaceuticallyacceptable carriers into unit dosage forms in a conventional manner sothat the patient in need of therapy for precancerous lesions canperiodically (e.g., once or more per day) take a compound according tothe methods of this invention. The exact initial dose of the compoundsof this invention can be determined with reasonable experimentation. Oneskilled in the art should understand that the initial dosage should besufficient to achieve a blood plasma concentration approaching apercentage of the IC₅₀ value of the compound, with the percentagedepending on the chemopreventative or chemotherapeutic indication. Theinitial dosage calculation would also take into consideration severalfactors, such as the formulation and mode of administration, e.g. oralor intravenous, of the particular compound. For example, assuming apatient with an average circulatory system volume of about four liters,based on the IC₅₀ values for compounds of this invention, one wouldcalculate a dosage of about 0.6 mg-4.0 gr of such compounds forintravenous administration to achieve a systemic circulatoryconcentration equivalent to the IC₅₀ concentration.

Compounds of this invention are also cGMP-specific PDE inhibitors astaught in U.S. patent application Ser. No. 09/046,739 filed Mar. 24,1998. Compounds can be tested for inhibitory effect on phosphodiesteraseactivity using either the enzyme isolated from any tumor cell line suchas HT-29 or SW-480. Phosphodiesterase activity can be determined usingmethods known in the art, such as a method using radioactive ³H cyclicGMP (cGMP)(cyclic 3′,5′-guanosine monophosphate) as the substrate forPDE5 enzyme. (Thompson, W. J., Teraski, W. L., Epstein, P. M., Strada,S. J., Advances in Cyclic Nucleotide Research, 10:69-92, 1979, which isincorporated herein by reference). In brief, a solution of definedsubstrate ³H-cGMP specific activity (0.2 μM; 100,000 cpm; containing 40mM Tris-HCl (pH 8.0), 5 mM MgCl₂ and 1 mg/ml BSA) is mixed with the drugto be tested in a total volume of 400 μl. The mixture is incubated at30° C. for 10 minutes with partially purified cGMP-specific PDE isolatedfrom HT-29 cells. Reactions are terminated, for example, by boiling thereaction mixture for 75 seconds. After cooling on ice, 100 μl of 0.5mg/ml snake venom (O. Hannah venom available from Sigma) is added andincubated for 10 min at 30° C. This reaction is then terminated by theaddition of an alcohol, e.g. 1 ml of 100% methanol. Assay samples areapplied to a anion chromatography column (1 ml Dowex, from Aldrich) andwashed with 1 ml of 100% methanol. The amount of radioactivity in thebreakthrough and the wash from the columns in then measured with ascintillation counter. The degree of PDE5 inhibition is determined bycalculating the amount of radioactivity in drug-treated reactions andcomparing against a control sample (a reaction mixture lacking thetested compound).

Using such protocols, the cGMP-specific PDE inhibitor of Example 1 hadan IC₅₀ value of 0.68 μM utilizing HT29 cell extracts.

It will be understood that various changes and modifications can be madein the details of procedure, formulation and use without departing fromthe spirit of the invention, especially as defined in the followingclaims.

We claim: 1.(Z)-5-Fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamide. 2.A method of preparing(Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamidehydrochloride, comprising: reacting(Z)-5-Fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)indenylacetamide withhydrochloric acid.